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EP1102838A2 - Procede de modulation de l'efficience de terminaison de translation et de la degradation de l'arnm aberrant faisant intervenir un complexe de surveillance comprenant la proteine humaine upf1p, le facteur 1 et le facteur 3 de liberation eucaryote - Google Patents

Procede de modulation de l'efficience de terminaison de translation et de la degradation de l'arnm aberrant faisant intervenir un complexe de surveillance comprenant la proteine humaine upf1p, le facteur 1 et le facteur 3 de liberation eucaryote

Info

Publication number
EP1102838A2
EP1102838A2 EP99953357A EP99953357A EP1102838A2 EP 1102838 A2 EP1102838 A2 EP 1102838A2 EP 99953357 A EP99953357 A EP 99953357A EP 99953357 A EP99953357 A EP 99953357A EP 1102838 A2 EP1102838 A2 EP 1102838A2
Authority
EP
European Patent Office
Prior art keywords
complex
erf3
upflp
erfl
translation termination
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP99953357A
Other languages
German (de)
English (en)
Other versions
EP1102838B1 (fr
Inventor
Stuart Peltz
Kevin Czaplinski
Youmin Weng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rutgers State University of New Jersey
Rutgers Health
Original Assignee
University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
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Publication of EP1102838A2 publication Critical patent/EP1102838A2/fr
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Publication of EP1102838B1 publication Critical patent/EP1102838B1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • the present invention relates to a multiprotein surveillance complex comprising human Upflp eucaryotic Release Factor 1 and eucaryotic Release Factor 3 which is involved in modulation of the efficiency of translation termination and degradation of aberrant mRNA.
  • Identification of this complex provides an in vitro assay system for identifying agents that: affect the functional activity of mRNAs by altering frameshift frequency; permit monitoring of a termination event; promote degradation of aberrant transcripts; provide modulators (inhibitors/stimulators) of peptidyl transferase activity during initiation, elongation, termination and mRNA degradation of translation.
  • Such agents which may be antagonists or agonists, are useful for screening, and diagnostic purposes, and as therapeutics for diseases or conditions which are a result of, or cause, premature translation.
  • NMD nonsense- mediated mRNA decay pathway
  • the proteins involved in promoting NMD have been investigated in C. elegans, mammalian cells and in the yeast Saccharomyces cerevisiae. Three factors involved in NMD have been identified in yeast. Mutations in the UPF1, UPF2, and UPF3 genes were shown to selectively stabilize mRNAs containing early nonsense mutations without affecting the decay rate of most wild-type mRNAs (He and Jacobson 1995, Lee and Culbertson 1995, Leeds et al. 1992, Leeds et al. 1991, Cui et al. 1995). Recent results indicate that the Upflp, Upf2p and Upf3 ⁇ interact and form a complex (He and Jacobson 1995, He et al. 1997, Weng et al. 1996b). In C.
  • a translation reinitiation event can prevent activation of the NMD pathway (Ruiz- Echevarria and Peltz, 1996; Ruiz-Echevarria et al., 1998; Zhang and Maquat, 1997). Taken together, these results indicate that the NMD pathway in yeast is a cytoplasmic and translation-dependent event.
  • the rent 1 /hup fl protein is also predominantly cytoplasmic (Applequist et al. 1997)
  • the yeast UPF1 gene and its protein product have been the most extensively investigated factor of the putative surveillance complex (Czaplinski et al. 1995, Weng et al. 1996a,b, Weng et al, 1998, Altamura et al. 1992, Cui et al. 1996, Koonin, 1992, Leeds et al. 1992, Atkin et al. 1995,1997).
  • the Upflp contains a cysteine- and histidine-rich region near its amino terminus and all the motifs required to be a member of the superfamily group I helicases.
  • the yeast Upflp has been purified and demonstrates RNA binding and RNA- dependent ATPase and RNA helicase activities (Czaplinski et al.
  • the ability to modulate translation termination has important implications for treating diseases associated with nonsense mutations. As with any biological system, there will be a small amount of suppression of a nonsense mutation, resulting in expression of a full length protein (which may or may not include an amino acid substitution or deletion). In the natural state, such low quantities of full length protein are produced that pathology results. However, by stabilizing the nonsense mRNA, the likelihood of "read-through" transcripts is dramatically increased, and may allow for enough expression of the protein to overcome the pathological phenotype.
  • the nonsense-mediated mRNA decay pathway is an example of an evolutionarily conserved surveillance pathway that rids the cell of transcripts that contain nonsense mutations.
  • the product of the UPF1 gene is a necessary component of the putative surveillance complex that recognizes and degrades aberrant mRNAs.
  • the results presented here demonstrate that the yeast and human forms of the Upflp interact with both eucaryotic translation termination factors eRFl and eRF3. Consistent with Upflp interacting with the eRFs, the Upflp is found in the prion-like aggregates that contain eRFl and eRF3 observed in yeast [PSP] strains. These results indicate that interaction of the Upflp with the peptidyl release factors is a key event in the assembly of the putative surveillance complex that enhances translation termination monitors whether termination has occurred prematurely and promotes degradation aberrant transcripts.
  • This invention provides an isolated complex comprising a human Upflp protein, a peptidyl eucaryotic release factor 1 (eRFl) and a peptidyl eucaryotic release factor 3 (eRF3), wherein the complex is effective to modulate peptidyl transferase activity.
  • this invention further comprises a human Up ⁇ p and Upf2p.
  • This invention provides an agent which binds to the complex comprising an amount of a human Upflp protein, a peptidyl eucaryotic release factor 1 (eRFl) and a peptidyl eucaryotic release factor 3 (eRF3) effective to modulate translation termination.
  • This invention provides an agent which binds to the complex of claim 1 , wherein the agent inhibits ATPase of Upflp; GTPase activity of eRFl or eRF3; or RNA binding to a ribosome.
  • This invention provides an agent which inhibits or modulates the binding of human Upflp to eRFl, or eRF3 or eRFl or eRF3 to Upflp.
  • This invention provides an agent which inhibits or modulates the binding of human Upf3p to eRF 1 , or eRF3 or eRF 1 or eRF3 to Upf3p.
  • This invention provides an agent which facilitates the binding of human Upflp to eRFl or eRF3; or eRF3 or eRFl or eRF3 to Upflp.
  • This invention provides an agent which facilitates the binding of human UpOp to eRFl or eRF3; or eRF3 or eRFl or eRF3 to Up ⁇ p.
  • This invention provides an agent which modulates the binding of human Upflp, eRFl or eRF3 to a ribosome.
  • This invention provides a method of modulating peptidyl transferase activity during translation, comprising contacting a cell with the complex in an amount effective to facilitate translation termination, thereby modulating the peptidyl transferase activity.
  • This invention provides a method of modulating peptidyl transferase activity during translation, comprising contacting a cell with the agent, in an amount effective to suppress non-sense translation termination, thereby modulating the peptidyl transferase activity.
  • the peptidyl transferase activity during translation occurs during initiation, elongation, termination and degradation of mRNA.
  • This invention provides a method of modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts, comprising contacting a cell with the agent, in an amount effective to inhibit the binding of human Upflp to eRFl, or eRF3; or eRFl or eRF3 to Upfl, thereby modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts.
  • This invention provides a method of modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts, comprising contacting a cell with an agent, which inhibits the ATPase/helicase activity of Upflp; the GTPase activity of eRFl or eRF3; or binding of RNA to a ribosome, thereby modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts.
  • This invention provides a method of screening for a drug involved in peptidyl transferase activity during translation comprising: a) contacting cells with a candidate drug; and b) assaying for modulation of the complex, wherein a drug that modulates the complex is involved in peptidyl transferase activity or enhancing translation termination.
  • This invention provides a method of screening for a drug involved in enhancing translation termination comprising: a) incubating the drug and the complex; and b) measuring the effect on non-sense suppression, thereby screening for a drug involved in enhancing translation termination.
  • the assays may be a RNA or NTPase assays, such as ATPase or GTPase.
  • This invention provides a method of modulating the efficiency of translation termination of mRNA and/or degradation of abberant transcripts in a cell, said method comprising: a) providing a cell containing a vector comprising the nucleic acid encoding proteins of the complex, the complex; or an antisense molecule thereof; b) overexpressing said nucleic acid in said cell to produce an overexpressed complex so as to interfere with the function of the complex.
  • This invention provides method for identifying a disease state involving a defect in the complex comprising: (a) transfecting a cell with a nucleic acid which encodes the complex; (b) determining the proportion of the defective complex of the cell after transfection; (c) comparing the proportion of the defective complex of the cell after transfection with the proportion of defective complex of the cell before transfection.
  • This invention provides a method for treating a disease associated with peptidyl transferase activity, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the complex or the agents, and a pharmaceutical carrier or diluent, thereby treating the subject.
  • FIGURE 1 The yeast Upfl protein interacts specifically with the peptidyl release factors.
  • GST-eRFl or GST-eRF3 fusion proteins bind specifically to Upflp in a yeast extract.
  • Cytoplasmic extracts from a yeast strain BJ3505 transformed with either pG-1 (vector) or pG-lFLAGUPFl (Flag-Upflp) were prepared in IBTB and incubated with 30 ⁇ l GST, GST-eRFl or
  • FIGURE 2 The Upflp is associated with eRF3 [PSP] aggregates. Cytoplasmic extracts from isogenic [PSP] and [psi -] variants of strain 7G-H66 upfl is. and containing FLAG-UPF1 inserted into a centromere plasmid were fractionated by centrifugation through a sucrose cushion as described previously (Paushkin et al. 1997b).
  • FIGURE 3 eRF3 and RNA compete for binding to Upflp.
  • A Poly(U) RNA prevents Upflp from binding to eRF3.
  • Reaction mixtures were prepared as described in Figure IB, except that binding was performed in TBSTB (TBST with 100 ⁇ g/ml BSA) and reaction mixtures contained lmM ATP, ImM GTP, or lOO ⁇ g/ml poly(U) RNA as indicated above each lane. The reaction mixtures were mixed for 1 hour at 4°C. Following mixing, the complexes were washed as in figure IB with TBSTB containing ImM
  • FIGURE 4 eRFl and eRF3 inhibit Upflp RNA-dependent ATPase activity.
  • Upflp RNA-dependent ATPase activity was determined in the presence of GST- RF fusions by a charcoal assay using 1 ⁇ g/ml poly(U) RNA with and lOO ⁇ g/ml BSA. The results are plotted as pmol of 32 P released versus the amount of the indicated protein.
  • FIGURE 5 A RENTl/HUPFl chimeric allele functions in translation termination.
  • a RENTl/HUPFl chimeric allele prevents nonsense suppression in a upfl A strain.
  • Strain PLY146 MAT ⁇ ura3-52 trpI ⁇ upfl:: URA3 leu2-2 tyr7-l
  • YCplac22 vector
  • YCpUPFl UPF1
  • YCpRentlCHI4-2 YEpRentlCHI4-2
  • RNA samples were prepared in -t ⁇ -met media and 5 ⁇ l of these dilutions were plated simultaneously on -t ⁇ -met (upper plate) or -t ⁇ -met-leu-tyr (lower plate) media. Cells were monitored for growth at 30°C.
  • YCplac22 (vector), YCpUPFl (UPF1), or YEpRentlCHI4-2 (YEpRENTlCHI4-2)(10) was subjected to northern blotting analysis and probed with either the LEU2, TYR7 or CYH2 probes.
  • FIGURE 6 Rentl /hupfl interacts with eRF 1 and eRF3.
  • Notl linearized pT7RE ⁇ T 1 (lanes 1-4) or luciferase template (lanes 5-8) was used in the TNT coupled Reticulocyte in vitro transcription translation as per manufacturers directions (Promega).
  • 2 ⁇ l of completed translation reactions were electrophoresed in lanes 1 and 5.
  • 5 ⁇ l of the completed reactions were incubated in 200 ⁇ l of IBTB with lO ⁇ l of GST, GST-eRFl or GST-eRF3 sepharose-protein complexes as indicated above each lane.
  • FIGURE 7 Model for Upfl function in mRNA surveillance.
  • A Modulation of RNA binding enhances interaction of Upfl with peptidyl release factors. ATP binding to Upflp decreases the affinity of Upfl for RNA. Since RNA and eRF3 compete for binding to Upfl, Interaction with eRF3 is favored.
  • B A model for mRNA surveillance. Interaction of Upflp with peptidyl release factors assembles an mRNA surveillance complex at a termination event. This interaction prevents Upfl from binding RNA and hydrolyzing
  • the release factors dissociate from the ribosome, activating the Upflp helicase activity.
  • the surveillance complex then scans 3' of the termination codon for a DSE. Interaction of the surveillance complex with the DSE signals that premature translation termination has occurred and the mRNA is then decapped and degraded by the Dcplp and Xrnlp exoribonuclease, respectively.
  • FIGURE 8 Schematic diagram of the vectors used to measure programmed -1 ribosomal frameshift efficiencies in vivo. Transcription is driven from the PGK1 promoter and uses the PGK1 translation initiation codon.
  • the bacterial lacZ gene In pTI25, the bacterial lacZ gene is in the 0-frame with respect to the start site. In plasmid pF8, the lacZ gene is positioned 3' of the L-A virus frameshift signal and in the -1 frame relative to the translation start site.
  • FIGURE 9 A up ⁇ strain increases programmed -1 ribosomal frameshifting independently of its ability to promote stabilization of nonsense- containing transcripts.
  • the abundance of the PGKl-LacZ -1 reporter mRNA in the different upf deletion strains was determined by RNase protection analysis.
  • the abundance of the U3 snRNA was used as an internal control for loading.
  • the abundance of the reporter transcript in the wild-type strain was taken arbitrarily as 1.0.
  • FIGURE 10 A up ⁇ A strain can not mantain the M, killer virus.
  • FIGURE 11 Paromomycin sensitivity was monitored in isogenic wild-type and up ⁇ A strains by placing a disc containing 1 mg of paromomycin onto a lawn of cells and determining the zone of growth inhibition around the disc.
  • the surveillance pathway eliminates aberrant mRNA that contains non-sense mutations with the protein coding region.
  • This invention is directed to three aspects of post-transcriptional regulation, including: suppression of nonsense mutations in inherited disease and cancers; inhibition of ribosomal frameshifting in viral infections; and alterations of RNA:protein interactions that, in turn, will modulate critical mRNA levels in multiple diseases.
  • the Upflp enhances translation termination by interacting with the peptidyl release factors eucaryotic Release Factor 1 (eRFl) and Release Factor 3 (eRF3) to augment their activity.
  • eRFl and eRF3 are conserved proteins that interact and promote peptidyl release in eucaryotic cells.
  • eRFl and eRF3 are encoded by the SUP45 and SUP 35 genes, respectively (Frolova et al. 1994, Zhouravleva et al. 1995). Sup45p and Sup35p have been shown to interact (Stansfield et al 1995, Paushkin et al 1997).
  • eRFl contains intrinsic peptide hydrolysis activity while eRF3, which has homology to the translation elongation factor EFl ⁇ (Didichenko et al. 1991), demonstrates GTPase activity (Frolova et al. 1996), and enhances the termination activity of eRFl (Zhouravleva et al. 1995).
  • the results presented herein demonstrate a biochemical interaction between the human and yeast Upflp and the peptidyl release factors eRFl and eRF3.
  • the following is a model for how the NMD pathway functions to enhance translation termination and subsequently recognize and degrade a nonsense-containing transcript.
  • a termination codon in the A site of a translating ribosome causes the ribosome to pause (Step 1).
  • the translation termination factors eRFl and eRF3 interact at the A site and promote assembly of the surveillance complex by interacting with Upflp, which is most likely complexed with other factors (Step 2).
  • the interaction of Upflp with the release factors inhibits its ATPase and RNA binding activities. This inhibition may be necessary in order for the Upflp to enhance the activity of the termination factors and ensure that the Upfp complex does not prematurely disassociate from release factors and search for a DSE.
  • Peptide hydrolysis occurs while the release factors are associated with the surveillance complex. Following GTP hydrolysis by eRF3 and completion of termination, the eRFs disassociate from the ribosome (Step 3). Disassociation of the release factors activates the RNA binding and ATPase activities of the Upflp and triggers the Upfp complex to scan 3' of the termination codon in search of a DSE (Step 4). If the complex becomes associated with the DSE or DSE-associated factors, an RNP complex forms such that the RNA is a substrate for rapid decapping by Dcplp (Step 5).
  • the RNP complex that forms as a consequence of the surveillance complex interacting with the DSE prevents the normal interaction between the 3' poly(A)-PABP complex and the 5' cap structure.
  • the uncapped mRNA is subsequently degraded by the Xrnlp exoribonuclease (Step 6).
  • a "surveillance complex” comprises at least Upflp; and eucaryotic Releasing Factor 1 and 3.
  • the "UPF1 " gene, is also called RENT1 or HUPF
  • the complex may also comprise Upf2p and /or Upf3p.
  • mRNA decay rates can be as short as 15-30 minutes or as long as 500 hours. Obviously, such differences in mRNA decay rates can lead to as much as 1000-fold differences in the level of specific proteins.
  • An additional level of control is provided by the observation that decay rates for individual mRNAs need not be fixed, but can be regulated as a consequence of autogenous feedback mechanisms, the presence of specific hormones, a particular stage of differentiation or the cell-cycle, or viral infection.
  • nonsense-mediated mRNA decay to human health is illustrated by the identification of a growing number of inherited disease in which nonsense mutations cause the disease state and in which nonsense mutations cause the disease state and in which the respective mRNAs have been shown to be substrates of the nonsense-mediated mRNA decay pathway.
  • This invention provides an isolated complex comprising a human Upflp protein, a peptidyl eucaryotic release factor 1 (eRFl) and a peptidyl eucaryotic release factor 3 (eRF3), wherein the complex is effective to modulate peptidyl transferase activity.
  • eRFl peptidyl eucaryotic release factor 1
  • eRF3 peptidyl eucaryotic release factor 3
  • Upflp interacts with the peptidyl release factors eRFl and eRF3: Upflp modulates translation termination by interacting with the peptidyl release factors eRFl and eRF3.
  • eRFl and eRF3 were individually expressed inE. coli as glutathione-S-transferase (GST) fusion proteins and purified using glutathione sepharose beads.
  • GST-RF (release factor) fusion proteins associated with the glutathione sepharose beads were added to a yeast cytoplasmic extract containing a FLAG epitope-tagged Upflp. Following incubation, the GST-RFs and associated proteins were purified by affinity chromatography and subjected to SDS-PAGE.
  • the complex further comprises human U ⁇ f3p.
  • the results presented here indicate that the Upf3p has a function in ensuring appropriate maintenance of translational reading frame.
  • the function of the Upf3p in this process appears to be genetically epistatic to the Upflp and Upf2p, since the programmed -1 frameshifting and killer maintenance phenotypes of a up ⁇ A are observed in upfl A and up ⁇ A strains.
  • the results presented here demonstrate that the Upfp's have distinct roles that can affect different aspects of the translation and mRNA turnover processes. Importantly these results may also have practical implications, since many viruses of clinical, veterinary and agricultural importance utilize programmed frameshifting.
  • programmed ribosomal frameshifting serves as a unique target for antiviral agents, and the identification and characterization of the factors involved in this process will help to develop assays to identify these compounds.
  • the complex comprises human Upf2p.
  • This invention provides an expression vector which comprises a nucleic acid encoding a human Upflp protein, a peptidyl eucaryotic release factor 1 (eRFl) and a peptidyl eucaryotic release factor 3 (eRF3) operably linked to a regulatory element.
  • eRFl peptidyl eucaryotic release factor 1
  • eRF3 peptidyl eucaryotic release factor 3
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e. , capable of replication under its own control.
  • a “cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites. The segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxy guanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester anologs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA -RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g. , restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e. , the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used.
  • vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g. , pGEX vectors, pmal-c, pFLAG, etc.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene are generated.
  • the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g. , E.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2 ⁇ plasmid.
  • Expression of DNA which encodes the proteins, Upflp, Upf2p,Up ⁇ p, and Release Factor 1 and 2 of the complex, i.e. may be controlled by any promoter /enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • Promoters which may be used are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
  • prokaryotic expression vectors such as the ⁇ -lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25).
  • Vectors are introduced into the desired host cells by methods known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g. , Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; Hartmut et al. , Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • This invention provides an agent which binds to the complex comprising an amount of a human Upflp protein, a peptidyl eucaryotic release factor 1 (eRFl) and a peptidyl eucaryotic release factor 3 (eRF3) effective to modulate translation termination.
  • This invention provides an agent which binds to the complex, wherein the agent inhibits ATPase of Upflp; GTPase activity of eRFl or eRF3; RNA binding; binding of the factors to the ribosome; or binding of the factors to each other.
  • This invention provides an agent which inhibits or modulates the binding of human Upflp to eRFl, or eRF3 or eRFl or eRF3 to Upflp; RNA binding; or binding of the factors to the ribosome; binding of the factors to each other.
  • This invention provides an agent which inhibits or modulates the binding of human Up ⁇ p to eRFl, or eRF3 or eRFl or eRF3 to Up ⁇ p.
  • This invention provides an agent which facilitates the binding of human Upflp to eRFl or eRF3; or eRF3 or eRFl or eRF3 to Upflp.
  • This invention provides an agent which facilitates the binding of human Up ⁇ p to eRFl or eRF3; or eRF3 or eRFl or eRF3 to Up ⁇ p; RNA binding; or binding of the factors to the ribosome; binding of the factors to each other.
  • This invention provides an agent which modulates the binding of human Upflp, eRFl or eRF3 to a ribosome.
  • the antibody may be a monoclonal or polyclonal antibody. Further, the antibody may be labeled with a detectable marker that is either a radioactive, colorimetric, fluorescent, or a luminescent marker.
  • the labeled antibody may be a polyclonal or monoclonal antibody. In one embodiment, the labeled antibody is a purified labeled antibody. Methods of labeling antibodies are well known in the art.
  • antibody includes, by way of example, both naturally occurring and non- naturally occurring antibodies. Specifically, the term “antibody” includes polyclonal and monoclonal antibodies, and fragments thereof. Furthermore, the term “antibody” includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. Further the protein or antibody may include a detectable marker, wherein the marker is a radioactive, colorimetric, fluorescent, or a luminescent marker.
  • Antibodies can be labeled for detection in vitro, e.g., with labels such as enzymes, fluorophores, chromophores, radioisotopes, dyes, colloidal gold, latex particles, and chemiluminescent agents.
  • the antibodies can be labeled for detection in vivo, e.g., with radioisotopes (preferably technetium or iodine); magnetic resonance shift reagents (such as gadolinium and manganese); or radio-opaque reagents.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the protein can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, "Co, 58 Co, 59 Fe, 90 Y, 125 1, 131 L and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • Complex specific antibodies and nucleic acids can be used as probes in methods to detect the presence of a complex polypeptide (using an antibody) or nucleic acid (using a nucleic acid probe) in a sample or specific cell type.
  • a complex -specific antibody or nucleic acid probe is contacted with a sample from a patient suspected of having a complex associated disorder, and specific binding of the antibody or nucleic acid probe to the sample detected.
  • the level of the complex or nucleic acid present in the suspect sample can be compared with the level in a control sample, e.g. , an equivalent sample from an unaffected individual to determine whether the patient has a complex -associated disorder.
  • Complex polypeptides, or fragments thereof can also be used as probes in diagnostic methods, for example, to detect the presence of complex -specific antibodies in samples. Additionally, complex -specific antibodies could be used to detect novel cofactors which have formed a complex with the complex or fragment thereof.
  • This invention provides a method of modulating peptidyl transferase activity during translation, comprising contacting a cell with the complex in an amount effective to facilitate translation termination, thereby modulating the peptidyl transferase activity.
  • This invention provides a method of modulating peptidyl transferase activity during translation, comprising contacting a cell with the agent, in an amount effective to suppress non-sense translation termination, thereby modulating the peptidyl transferase activity.
  • the peptidyl transferase activity during translation occurs during initiation, elongation, termination and degradation of mRNA.
  • This invention provides a method of modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts, comprising contacting a cell with the agent, in an amount effective to inhibit the binding of human Upflp to eRFl, or eRF3; or eRFl or eRF3 to Upfl, thereby modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts.
  • This invention provides a method of modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts, comprising contacting a cell with an agent, which inhibits the ATPase/helicase activity of Up ⁇ l; the GTPase activity of eRFl or eRF3; RNA binding; or binding of RNA to a ribosome, thereby modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts
  • agents that interfere with NTPase activity such as, ATPase activity, GTPase, helicase activity, or zinc finger motif configuration may be selected for testing.
  • agents may be useful drugs for treating viral infections, since many retroviruses, notably HIV, coronaviruses, and other RNA viruses that are associated with medical and veterinary pathologies.
  • an initial screen for agents may include a binding assay to such proteins. This assay may be employed for testing the effectiveness of agents on the activity of frameshift associated proteins from human as well as yeast or other non- human source, including but not limited to animals.
  • Antisense RNAs are small, diffusible, untranslated and highly structured transcripts that pair to specific target RNAs at regions of complementarity, thereby controlling target RNA function or expression.
  • attempts to apply antisense RNA technology have met with limited success. The limiting factor appears to be in achieving sufficient concentrations of the antisense RNA in a cell to inhibit or reduce the expression of the target gene.
  • the agents of the invention that stabilize aberrant mRNA transcripts may also stabilize antisense RNAs.
  • Presence, relative abundance, or absence of the complex is determined by the binding of the antibody. Possible detection methods including affinity chromatography, Western blotting, or other techniques well known to those of ordinary skill in the art.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (see Marcus-Sekura, 1988, Anal. Biochem. 172:298). In the cell, they hybridize to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein.
  • Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into organ cells.
  • Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988, supra; Hambor et al., 1988, J. Exp. Med. 168:1237).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns . By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J. Am. Med. Assoc. 260:3030). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead "-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target MRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species, and eighteen base recognition sequences are preferable to shorter recognition sequences.
  • This invention provides a method of screening for a drug involved in peptidyl transferase activity during translation comprising: a) contacting cells with a candidate drug; and b) assaying for modulation of the complex, wherein a drug that modulates complex is involved in peptidyl transferase activity.
  • the complex may be assayed for NTPase activity, such as ATPase, GTPase, RNA binding acitivty, factors which bind to the complex, such as but not limited to eRFl and eRF3, factors which dissociate from the ribosome, factors which promote aggregation; factors which enhance translation termination by slowing peptide hydrolysis.
  • This invention provides a method of screening for a drug active involved in enhancing translation termination comprising: a) contacting cells with a candidate drug; and b) assaying for modulation of the protein complex; wherein a drug that modulates protein complex is involved in enhancing translation termination.
  • This invention provides a method of screening for a drug involved in enhancing translation termination comprising: a) incubating the drug and the complex; and b) measuring the effect on non-sense suppression, thereby screening for a drug involved in enhancing translation termination.
  • the assays may be a RNA or NTPase assays, such as ATPase, or GTPase assays which are known to those skilled in the art.
  • the presence, relative abundance of, or absence of the complex may be detected by binding to an antibody.
  • Upfl may be detected using the M2 mouse monoclonal antibody against the FLAG epitope as described previously (Czaplinski et al. 1995, Weng et al. 1996a,b).
  • eRF3 was detected as described in Didichenko et al. 1991.
  • eRFl was detected as described in Stansfield et al. 1992.
  • Upflp RNA-dependent ATPase activity may be determined using 20 ng Upflp in the presence of GST-RF fusion proteins by a charcoal assay as described previously (Czaplinski et al. 1995) using 1 ⁇ g/ml poly(U) RNA with and lOO ⁇ g/ml BSA.
  • RNA binding may be determined as follows: A uniformly labeled 32 nt RNA was synthesized by SP6 transcription of Sstl digested pGEM5Zf(+) as described previously (Czaplinski et al. 1995). RNA binding buffer was as described previously (Czaplinski et al. 1995) with the exception that lOO ⁇ g/ml BSA was included in all reactions. The indicated amounts of GST-eRF3 (28), were incubated with 200ng Upflp for 15 minutes at 4°C. 50 fmol of the RNA substrate was added and incubated for 5 minutes. Stop solution was added, and reactions electrophoresed in a 4.5% native PAGE gel (0.5xTBE, 30:0.5 acrylamide:bisacrylamide, with 5% glycerol).
  • This invention provides a method of modulating the efficiency of translation termination of mRNA and/or degradation of abberant transcripts in a cell, said method comprising: a) providing a cell containing a vector comprising the nucleic acid encoding the complex; or an antisense thereof; b) overexpressing said nucleic acid vector in said cell to produce an overexpressed complex so as to interfere or inhibit with the function of the complex.
  • This invention provides method for identifying a disease state involving a defect in the complex of claim 1 comprising: (a) transfecting a cell with a nucleic acid which encodes the complex; (b) determining the proportion of the defective complex of the cell after transfection; (c) comparing the proportion of the defective complex of the cell after transfection with the proportion of defective complex of the cell before transfection.
  • nonsense-mediated mRNA decay leads to cellular deficiencies of essential proteins and hence to disease. Altered control of the stability of normal mRNAs can have comparably dire consequences.
  • This invention provides a method for treating a disease associated with peptidyl transferase activity, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the complex of claim 1 or the agents which modulate or stimulate the complex, and a pharmaceutical carrier or diluent, thereby treating the subject.
  • Nonsense mutations cause approximately 20-40% of the individual causes of over 240 different inherited diseases (including cystic fibrosis, hemophilia, familial hypercholesterolemia, retinitis pigmentosa, Duchenne muscular dystrophy, and Marfan syndrome). For many diseases in which only one percent of the functional protein is produced, patients suffer serious disease symptoms, whereas boosting expression to only five percent of normal levels can greatly reduce the severity or eliminate the disease.
  • the disease, proteins, or genes which are as a result of non-sense or frameshift mutations include but are not limited to the following: HEMOGLOBIN-BETA LOCUS; CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR; MUSCULAR DYSTROPHY, PSEUDOHYPERTROPHIC PROGRESSIVE, DUCHENNE AND BECKER YPES; PHENYLKETONURIA, INSULIN RECEPTOR; HEMOPHILIA A, ADENOMATOUS POLYPOSIS OF THE COLON, HYPERCHOLESTEROLEMIA, FAMILIAL, NEUROFIBROMATOSIS, TYPE I, HEMOPHILIA B, HYPERLIPOPROTEINEMIA TYPE I, TAY-SACHS DISEASE, BREAST CANCER TYPE 1, ADRENAL HYPERPLASIA, VON WILLEBRAND DISEASE, MUCOPOLYSACCHARIDOSIS TYPE I, ALBINI
  • CEREVISIAE EXCISION-REPAIR CROSS-COMPLEMENTING RODENT REPAIR DEFICIENCY
  • GROUP 6 MAPLE SYRUP URINE DISEASE APOPTOSIS ANTIGEN 1, TRANSCRIPTION FACTOR 1, HEPATIC, UBIQUITIN-PROTELN LIGASE E3A, TRANSGLUTAMLNASE 1, MYOSLN VILA, GAP JUNCTION PROTEIN, BETA-1, 32-KD, TRANSCRIPTION FACTOR 2, HEPATIC, PROTEIN 4.2, ERYTHROCYTIC, THYROID-STIMULATING HORMONE, BETA CHAIN, TREACHER COLLLNS-FRANCESCHETTI SYNDROME 1, CHOROLDEREMIA, ENDOCARDIAL FIBROELASTOSIS-2, COWDEN DISEASE, ANTI-MULLERIAN HORMONE, SRY-BOX 10, PTA DEFICIENCY TYROSLNASE-RELATED PROTE
  • MUCOPOLYSACCHARIDOSIS TYPE IIIB P-GLYCOPROTELN-3 SEVERE COMBINED IMMUNODEFICIENCY, B -CELL-NEGATIVE RETINITIS PIGMENTOSA, RIBOSOMAL PROTEIN S6 KINASE, 90 KD, POLYPEPTIDE 3, SYNDROME SYNDROME, FACTOR DEFICIENCY X-LINKED, AGAINST DECAPENTAPLEGIC, DROSOPHILA, HOMOLOG OF, 4, FACTOR FOR COMPLEMENT, DEHYDROGENASE/DELTA-ISOMERASE, TYPE I CONDUCTIVE, WITH STAPES FIXATION AQP1 1, PROGRESSIVE, PROGRESSIVE FAMILIAL INTRAHEPATIC, TYPE III MONOPHOSPHATE DEAMLNASE-1, HOMEO BOX TRANSCRIPTION FACTOR 1.
  • This invention provides methods to screen drugs which acts as therapeutics that treat diseases caused by nonsense and frameshift mutations.
  • biochemical and in vitro assays which monitor the activity of ATP binding, ATPase activity, RNA helicase activity, GTP binding, GTPase activity, release factors, or RNA binding to the complex or to each other (i.e. Upflp to eRFl and eRF3, or Up 2 to Up ⁇ p ); developing assays capable of quantitating the activity of the human gene product in mRNA decay and translational suppression; screening compounds using aforementioned assays.
  • test composition is any composition such as a gene, a nucleic acid sequence, a polypeptide, peptide fragment or composition created through the use of a combinatorial library or other combinatorial process that can be assayed for its ability to function in given capacity or compound which mimics the activity of the complex. Often such a test composition, nucleic acid sequence or polypeptide is, because of its sequence or structure, suspected of being able to function in a given capacity.
  • a “co-factor” is any composition (e.g., a polypeptide, polypeptide derivative, or peptidomimetic) that is capable of modulating the complex and influencing NMRD or efficiency of translation termination. Included are compositions that naturally induce NMRD or the efficiency of translation termination via the complex; also included are compositions that do not naturally induce NMRD (e.g., artificial compositions and natural compositions that serve other purposes).
  • the term "agonist” as used herein means any composition that is capable of increasing or stimulating the efficiency of translation termination or mRNA degredation by interacting with or binding to the complex or factors, such as eRFl or eR ⁇ , of the complex which interact with Upflp of the complex..
  • antagonist means any composition that is capable of decreasing or inhibiting the efficiency of translation termination or mRNA degredation by interacting with or binding to the complex or factors, such as eRFl or eR ⁇ , of the complex which interact with Upflp of the complex.
  • the invention also provides a method for determining whether a test agent or composition modulates the complex in a cell.
  • the method can be performed by (i) providing a cell that has the complex; (ii) contacting the cell with a test agent or composition that, in the absence of the test agent or composition, activates the complex in the cell; and (iii) detecting a change in the complex of the cell.
  • the cell can be contacted with the test agent or composition either simultaneously or sequentially.
  • An increase in the complex indicates that the test agent or composition is an agonist of the complex while a decrease in the complex indicates that the test agent or composition is an antagonist of the complex.
  • the above-described method for identifying modulators of the complex can be used to identify compositions, co-factors or other compositions within the complex pathway comprising the complex for use in this aspect of the invention.
  • Any agent or composition can be used as a test agent or composition in practicing the invention; a preferred test agent or compositions include polypeptides and small organic agent or compositions.
  • sequence or structural homology can provide a basis for suspecting that a test agent or composition can modulate the complex in a cell, randomly chosen test agent or compositions also are suitable for use in the invention.
  • Art-known methods for randomly generating an agent or compositions e.g. , expression of polypeptides from nucleic acid libraries
  • Those skilled in the art will recognize alternative techniques can be used in lieu of the particular techniques described herein.
  • the invention also provides a method for detecting novel co-factors or inhibitors which bind the complex which comprises contacting a sample comprising the complex with test compositions and measuring the change in the complex after application of the test composition.
  • the complex of the instant invention is useful in a screening method for identifying novel test compounds or novel test compositions which affect the complex.
  • the invention provides a method for screening test compositions comprising incubating components, which include the test composition, and the complex under conditions sufficient to allow the components to interact, then subsequently measuring the effect the test composition has on the complex in a test cell.
  • the observed effect on the complex and a composition may be either agonistic or antagonistic.
  • This invention provides a method for identifying a disease state involving defective the protein complex comprising: (a) transfecting a cell with a nucleic acid which encodes the protein complex; (b) determining the proportion of the defective protein complex of the cell after transfection; (c) comparing the proportion of the defective protein complex of the cell after transfection with the proportion of defective protein complex of the cell before transfection.
  • Any screening technique known in the art can be used to screen for agents that affect translation termination or a mRNA decay protein.
  • the present invention contemplates screens for small molecule ligands.
  • the screening can be performed with recombinant cells that express the proteins, complexes involved in translation termination or mRNA decay protein, or alternatively, with the purified protein.
  • the ability of labeled protein to bind to a molecule in a combinatorial library can be used as a screening assay, as described in the foregoing references.
  • This invention provides a method of screening a candidate host cell for the amount of the complex produced by said cell relative to a control cell, said method comprising: a) providing a clonal population of said candidate host cell; b) treating said clonal population of cells such that the intracellular proteins are accessible to an antibody; c) contacting said intracellular proteins with an antibody that specifically binds to the complex; and d) determining the relative amount of the complex produced by said candidate host cell.
  • This invention provides a method of substantially inhibiting translation termination efficiency of mRNA and/or degradation of aberrant transcripts in a cell, said method comprising: a) providing a cell containing the DNA; b) overexpressing said DNA in said cell to produce an overexpressed polypeptide that binds to Upflp and interferes with Upflp function.
  • This invention provides a method of substantially inhibiting translation termination efficiency of mRNA and/or degradation of aberrant transcripts in a cell in a cell, said method comprising: a) providing a cell; b) expressing antisense transcript of the complex in sufficient amount to bind to the complex.
  • This invention provides a method of substantially inhibiting translation termination in a cell, said method comprising: mutating the complex comprising Upflp, Upf2p, Up ⁇ p, eRFl, and eRF3, such that essentially no functional complex is produced in said cell.
  • This invention provides a method for treating a disease associated with translation termination efficiency of mRNA and/or degradation of aberrant transcripts, comprising administering to a subject administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the complex which is introduced into a cell of a subject; and a pharmaceutical carrier or diluent, thereby treating the subject.
  • the invention provides a method of treating a patient having or at risk of having early stage as a result of genetic deficiency, disease or clinical treatment wherein the condition has an etiology associated with a defective, the method comprising administering to the patient a therapeutically effective amount of a formulation or composition which modulates the expression of the complex such that the state of the patient is ameliorated.
  • “Therapeutically effective” as used herein refers to an amount formulation that is of sufficient quantity to ameliorate the state of the patient so treated.
  • “Ameliorate” refers to a lessening of the detrimental effect of the disease state or disorder in the patient receiving the therapy.
  • the subject of the invention is preferably a human, however, it can be envisioned that any animal can be treated in the method of the instant invention.
  • modulate means enhance, inhibit, alter, or modify the expression of the complex, mRNA, nucleic acid, polypeptide or protein.
  • the present invention provides a number of routes for affecting translation termination, which has important implications for antiviral therapy and for suppression of pathological nonsense mutations.
  • the present invention provides drugs for use as antiviral compounds or to alter ribosomal decay.
  • drug is used herein to refer to a compound or agents, such as an antibiotic or protein, that can affect function of the peptidyl transferase center during initiation, elongation, termination, mRNA degredation. Such compounds can increase or decrease aberrant mRNA and the efficiency of translation termination.
  • a nucleic acid encoding the complex or factors of the complex; an antisense or ribozyme specific for the complex, or specific for regions of the release factors and Upflp are introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell.
  • adipose tissue can be specifically targeted.
  • particular vectors include, but are not limited to , a defective herpes virus 1 (HSV1) vector [Kaplitt et al., Molec. Cell. Neurosci. 2:320- 330 (1991)], an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. [J. Clin. Invest. 90:626-630 (1992)], and a defective adeno- associated virus vector [Samulski et al. , J. Virol. 61:3096-3101 (1987); Samulski et al., J. Virol. 63:3822-3828 (1989)].
  • HSV1 herpes virus 1
  • the gene can be introduced in a retroviral vector, e.g. , as described in Anderson et al. , U.S. Patent No. 5,399,346; Mann et al., 1983, Cell
  • the vector can be introduced in vivo by lipofection.
  • liposomes for encapsulation and transfection of nucleic acids in vitro.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner, et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031 (1988)].
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes [Feigner and Ringold, Science 337:387-388 (1989)].
  • lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as pancrease, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting [see Mackey, et. al., supra]. Targeted peptides, e.g. , hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter [see, e.g. , Wu et al., J. Biol. Chem. 267:963-967 (1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990].
  • the present invention provides for co-expression of a gene product that modulates activity at the peptidyl transferase center and a therapeutic heterologous antisense or ribozyme gene under control of the specific DNA recognition sequence by providing a gene therapy expression vector comprising both a gene coding for a modulator of a peptidyl transferase center (including but not limited to a gene fo r a mutant frameshift or mRNA decay protein, or an antisense RNA or ribozyme specific for mRNA encoding such a protein) with a gene for an unrelated antisense nucleic acid or ribozyme under coordinated expression control.
  • these elements are provided on separate vectors; alternatively these elements may be provided in a single expression vector.
  • the present invention provides the means to treat viral infections by providing agents that modulate translation termination, and thus directly affect viral replication or assembly of viral particles.
  • the present invention advantageously provides drugs and methods to identify drugs for use in antiviral (or nonsense suppression) therapy of viruses that use the basic -1 ribosomal frameshifting mechanism, which includes four large families of animal viruses and three large families of plant viruses.
  • this invention provides assays for screening agents, antagonist/agonists, which effect frameshifting involving the complex, and which involve Up ⁇ p. Also, this invention provides a mutant Up ⁇ .
  • retroviruses use -1 ribosomal frameshifting, including lentiviruses (immunodeficiency viruses) such as HIV-1 and HIV-2, SIV, FIV, BIV, Visna virus, Arthritis-encephalitis virus, and equine infectious anemia virus; spumaviruses (the foamy viruses), such as human foamy virus and other mammalian foamy viruses; the T cell lymphotrophic viruses, such as HTLV-I, HTLV-II, STLVs, and BLV; avian leukosis viruses, such as leukemia and sarcoma viruses of many birds, including commercial poultry; type B retroviruses, including mouse mammary tumor virus; and type D retroviruses, such as Mason-Pfizer monkey virus and ovine pulmonary adenocarcinoma virus.
  • coronaviruses use the -1 frameshifting, including human coronaviruses, such as 229-E, OC43; animal coronaviruses, such as calf coronavirus, transmissible gastroenteritis virus of swine, hemagglutinating encephalomyelitis virus of swine, and porcine epidemic diarrhea virus; canine coronavirus; feline infectious peritonitis virus and feline enteric coronavirus; infectious bronchitis virus of fowl and turkey bluecomb virus; mouse hepatitis virus, rat coronavirus, and rabbit coronavirus.
  • human coronaviruses such as 229-E, OC43
  • animal coronaviruses such as calf coronavirus, transmissible gastroenteritis virus of swine, hemagglutinating encephalomyelitis virus of swine, and porcine epidemic diarrhea virus
  • canine coronavirus feline infectious peritonitis
  • torovirus a type of coronavirus
  • human toroviruses associated with enteric and respiratory diseases
  • breda virus of calves and bovine respiratory virus breda virus of calves and bovine respiratory virus
  • berne virus of horses porcine torovirus
  • feline torovirus feline torovirus.
  • Another coronavirus is the arterivirus, which includes simian hemorrhagic fever virus, equine arteritis virus, Lelystad virus (swine), VR2332 virus (swine), and lactate dehydrogenase-elevating virus (rodents).
  • animal viruses are paramyxoviruses, such as human -1 ribosomal frameshifting reported in measles, and astro viruses, such as human astroviruses 1-5, and bovine, ovine, porcine, canine, and duck astroviruses.
  • the plant viruses that involve a -1 frameshifting mechanism include tetraviruses, such as sobemoviruses (e.g. , southern bean mosaic virus, cocksfoot mettle virus), leuteoviruses (e.g. , barley yellowswarf virus, beet western yellows virus, and potato leaf roll virus), enamoviruses (e.g. , pea mosaic virus), and umbraviruses (e.g. , carrot mottle virus); tombusviruses, such as tombusvirus (e.g. , tomato bushy stunt virus), carmovirus (e.g. , carnation mottle virus), necrovirus (e.g. , tobacco necrosis virus); dianthoviruses (e.g. , red clover necrotic mosaic virus), and machiomovirus (e.g. , maize chlorotic mottle virus).
  • sobemoviruses e.g. , southern bean mosaic virus, cocksfoot
  • totiviruses such as L-A and L-BC (yeast) and other fungal viruses, giradia lamblia virus (intestinal parasite), triconella vaginell virus (human parasite), leishmania brasiliensis virus (human parasite), and other protozoan viruses are -1 frameshift viruses.
  • the component or components of a therapeutic composition of the invention may be introduced or administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermaly, subcutaneously, intraperitonealy, intraventricularly, or intracranialy.
  • Modes of delivery include but are not limited to: naked DNA, protein, peptide, or within a viral vector, or within a liposome.
  • the viral vector is a retro virus, adeno-associated virus, or adenovirus.
  • compositions and methods of the present invention are particularly suited to treatment of any animal, particularly a mammal, more specifically human.
  • domestic animals such as feline or canine subjects
  • farm animals such as but not limited to bovine, equine, caprine, ovine, and porcine subjects
  • wild animals whether in the wild or in a zoological garden
  • research animals such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e. , for veterinary medical use.
  • pharmaceutical composition could mean therapeutically effective amounts of the complex with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers useful in SCF therapy.
  • a “therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen.
  • compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris- HCL, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent abso ⁇ tion to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or inco ⁇ oration of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, pol
  • compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of SCF.
  • the choice of compositions will depend on the physical and chemical properties of the protein having SCF activity. For example, a product derived from a membrane-bound form of SCF may require a formulation containing detergent.
  • Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
  • particulate compositions coated with polymers e.g., poloxamers or poloxamines
  • SCF coupled to antibodies directed against tissue- specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.
  • Other embodiments of the compositions of the invention inco ⁇ orate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.
  • pharmaceutically acceptable carrier include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline.
  • pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host.
  • the amount of the compound may vary depending on its specific activity and suitable dosage amounts may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. In one embodiment the amount is in the range of 10 picograms per kg to 20 milligrams per kg. In another embodiment the amount is 10 picograms per kg to 2 milligrams per kg. In another embodiment the amount is 2-80 micrograms per kilogram. In another embodiment the amount is 5-20 micrograms per kg.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • the therapeutic compound can be delivered in a controlled release system.
  • the complex may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • a controlled release device is introduced into a subject in proximity of the site of inappropriate immune activation or a tumor.
  • Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
  • the methods and pharmaceutical compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e., for veterinary medical use.
  • domestic animals such as feline or canine subjects
  • farm animals such as but not limited to bovine, equine, caprine, ovine, and porcine subjects
  • wild animals whether in the wild or in a zoological garden
  • research animals such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e., for veterinary medical use.
  • EXAMPLE 1 Enhancement of Translation Termination And Degradation of Aberrant mRNAs
  • the nonsense-mediated mRNA decay pathway is an example of an evolutionarily conserved surveillance pathway that rids the cell of transcripts that contain nonsense mutations.
  • the product of the UPF1 gene is a necessary component of the putative surveillance complex that recognizes and degrades aberrant mRNAs.
  • the results presented here demonstrate that the yeast and human forms of the Upflp interact with both eucaryotic translation termination factors eRFl and eRF3. Consistent with Upflp interacting with the eRFs, the Upflp is found in the prion-like aggregates that contain eRFl and eRF3 observed in yeast [PSP] strains. These results indicate that interaction of the Upflp with the peptidyl release factors is a key event in the assembly of the putative surveillance complex that enhances translation termination monitors whether termination has occurred prematurely and promotes degradation aberrant transcripts.
  • Yeast Media was prepared as described (Rose et al. 1990). Yeast Transformations were performed by the lithium acetate method (Scheistl and Geitz 1989). RNA isolation, blotting and hybridization was as described (Weng et al. 1996a, Hagan et al. 1995).
  • Plasmid YCp and YEp RENTCHI4-2 were created by ligating a 4.5kb Sstl- Asp718 fragment from pMET25CHLMERA (Perlick et al. 1996) harboring the chimeric gene under the MET25 promoter into YCplac22 and YEplacl 12 (Ferguson et al. 1981) respectively.
  • YCpFLAGUPFl and YEpFLAGUPFl were described previously (Weng et al. 1996a).
  • GST-RF fusion plasmids, pGEX2T, pGEX2T-SUP35 and pGEX2T-SUP45 were described previously (Paushkin et al. 1997b).
  • Sepharose beads were collected at 500xg for 3 minutes, washed for 3 minutes with TBST supplemented with NaCl to 500mM and collected as before for a total of 2 times.
  • the Sepharose-protein complexes were then washed and collected as before with IBTB (25mM Tris-HCl pH 7.5, 50mM KC1, lOmM MgCl 2 , 2% glycerol, 0.1% Triton x-100, lOO ⁇ g/ml BSA) for a total of 2 times, and resuspended in IBTB to yield a 2:1 ratio of buffer to packed bead volume, l ⁇ l of GST-RF complexes typically contained 0.9 ⁇ g GST-eRFl or 1.5 ⁇ g GST-eRF3, while GST complexes typically contained 4.5 ⁇ g GST per ⁇ l of resin.
  • IBTB 25mM Tris-HCl pH 7.5, 50mM KC1, lOmM MgCl 2 ,
  • PI protease inhibitors
  • GST fusion proteins were eluted by resuspending the washed sepharose beads in 400 ⁇ l glutathione elution buffer (lOmM Tris-HCl pH8.0, ImM glutathione) and incubating at room temperature for 10 minutes with mixing. Sepharose beads were collected and the supernatant removed. Elution was repeated as before for a total of 3 times, and the elution fractions combined. Concentration of proteins was determined by the Bradford assay.
  • RNA-dependent ATPase activity was determined using 20 ng Upflp in the presence of GST-RF fusion proteins by a charcoal assay as described previously (Czaplinski et al. 1995) using 1 ⁇ g/ml poly(U) RNA with and lOO ⁇ g/ml BSA. The results are plotted as pmol of 32 P released versus the concentration of the indicated protein.
  • RNA binding assay A uniformly labeled 32 nt RNA was synthesized by SP6 transcription of Sstl digested pGEM5Zf(+) as described previously (Czaplinski et al. 1995). RNA binding buffer was as described previously (Czaplinski et al. 1995) with the exception that lOO ⁇ g/ml BSA was included in all reactions. The indicated amounts of GST-eRF3 (28), were incubated with 200ng Upflp for 15 minutes at 4°C. 50 fmol of the RNA substrate was added and incubated for 5 minutes. Stop solution was added, and reactions electrophoresed in a 4.5% native PAGE gel (0.5xTBE, 30:0.5 acrylamide:bisacrylamide, with 5% glycerol).
  • Upflp interacts with the peptidyl release factors eRFl and eRF3: Upflp modulates translation termination by interacting with the peptidyl release factors eRFl and eRF3.
  • eRFl and eRF3 were individually expressed inE. coli as glutathione-S-transferase (GST) fusion proteins and purified using glutathione sepharose beads.
  • GST-RF (release factor) fusion proteins associated with the glutathione sepharose beads were added to a yeast cytoplasmic extract containing a FLAG epitope-tagged Upflp (Czaplinski et al. 1995, Weng et al. 1996a,b).
  • the GST-RFs and associated proteins were purified by affinity chromatography and subjected to SDS- PAG ⁇ . Immunoblotting was performed and the presence of the Upflp was assayed using an antibody against the FLAG epitope.
  • the anti-FLAG antibody recognized only the 109 kD Upflp in cytoplasmic extracts from cells transformed with plasmid expressing the FLAG-Upflp (Fig. 1A, compare lane 2 to lane 1).
  • This analysis also demonstrated that the Upflp specifically co-purified with either eRFl (Fig. 1 A, lane 5) or eRF3 (Fig. 1 A, lane 4).
  • Upflp did not co-purify with GST protein that was not fused to another protein (Fig. 1 A, lane 3) or a GST-JIP protein, in which a Jak2 interacting protein fused to GST was used to monitor the specificity of the reaction.
  • the Upflp is associated with the aggregates of eRF3 in [PSP] strains:
  • the biochemical results demonstrated that the Upflp could enhance translation termination at a nonsense codon by interacting with the peptidyl release factors and enhancing their activity.
  • Recent results have shown that the nonsense suppressor phenotype observed in strains carrying the cytoplasmically-inherited determinant [PSP] is a consequence of a specific alternative protein conformational state of the yeast eRF3 (Sup35p).
  • eRF3 forms high-molecular weight aggregates, or an amyloid-like fiber, which inhibit eRF3 activity, leading to increased readthrough of translation termination codons by ribosomes (Wickner, 1994; Paushkin et al. 1997a, Patino et al. 1996; Glover et al., 1997). It was also suggested that this specific alternative conformation of eRF3 is capable of self-propagation by an autocatalytic mechanism, analogous to that of mammalian prions (Paushkin et al. 1997a, Glover et al. 1997, Wickner, 1994).
  • the alternative protein conformational state of the eRF3, and not a mutation in the SUP 35 gene allows self-propagation of the [PSP] phenotype.
  • Yeast eRFl (Sup45p) interacts with eRF3 and was also found in the aggregates present in [PSP] cells (Paushkin et al. 1997b).
  • RNA and eRF3 compete for binding to Upflp.
  • the effect of eRF3 on the ability of Upflp to complex with RNA was monitored.
  • Reaction mixtures containing Upflp and RNA, and either lacking or containing increasing concentrations of eRF3, were prepared and the formation of the Upflp:RNA complex was monitored by an RNA gel shift assay (Czaplinski et al. 1995, Weng et al. 1996a,b, Weng et al. 1998).
  • Fig. 3C, lane 2 increasing concentrations of eRF3 in the reaction mixtures reduced the amount of the Upflp-RNA complex that formed (Fig. 3C, lane 4-8).
  • the K436A form of the Upflp demonstrates altered interactions with the translation termination release factors: It was next determined whether a mutation in the UPFl gene that inactivated its mRNA turnover and translation termination activities affected the ability of the Upflp to interact with the translation termination release factors. Previous results have shown that strains harboring mutations in the conserved lysine residue in position 436 of the Upflp (K436) result in stabilization of nonsense- containing mRNAs and a nonsense suppression phenotype (Weng et al., 1996a). Using a purified K436A form of the Upflp (Weng et al, 1996a,1998), it was questioned whether this mutation affected the ability of the Upflp to interact with the eRFl.
  • the ability of the K436A Upflp to interact with eRF3 was monitered.
  • a reaction mixture containing the K436A Upflp and GST-eRF3 was prepared and the Upflp-eRF3 interaction was monitored as described above.
  • the K436A mutation affected the ability of the Upflp to preferentially interact with eRF3 versus RNA when ATP is present in the reaction mixture.
  • the K436A mutation has been shown to reduce the affinity of the Upflp for ATP (Weng et al., 1996a, 1998).
  • eRFl and eRF3 inhibit Upflp ATPase activity :
  • the genetic and biochemical data indicated that the ATPase/helicase activities were not required for enhancing translation termination but were necessary to degrade nonsense-containing transcripts (Weng et al., 1996a,b; Weng et al., 1997).
  • the interaction of the Upflp with the eRFs was predicted to inhibit its ATPase/helicase activity, thus allowing the Upflp to enhance translation termination. Therefore, the interaction of Upflp with either eRFl or eRF3 was examined if it would affect the RNA-dependent ATPase activity of Upflp.
  • Reaction mixtures were prepared containing radiolabeled ⁇ 32 P-ATP and either 1) Upflp, 2) Upflp and RNA, 3) Upflp, RNA and GST, 4) Upflp, RNA and GST-eRFl or 5) Upflp, RNA and GST-eRF3.
  • the ATPase activity in these reactions was monitored using a charcoal assay as described previously (Czaplinski et al. 1995, Weng et al. 1996a, Weng et al. 1996b). The results demonstrated that reactions containing only Upflp had no detectable ATPase activity while reactions containing Upflp and poly(U) RNA demonstrated maximal ATPase activity .
  • the rentl/hupfl contains both the cysteine/histidine-rich region and helicase motifs found in the yeast UPFl gene and displays 60% identity and 90% similarity over this region (Perlick et al. 1996, Applequist et al. 1997).
  • the hybrid construct used in these experiments consisted of the conserved domains from the human protein sandwiched between the N and C termini from the yeast UPFl gene (Perlick et al. 1996). This hybrid gene was previously shown to complement a upfl A strain in a frameshift allosuppression assay (Perlick et al. 1996). It was initially asked whether expression of the hybrid gene would function to prevent nonsense suppression.
  • a upfl A strain harboring leu2-2 and tyr 7-7 nonsense alleles was transformed with plasmids harboring either; 1) the vector alone, 2) the wild-type yeast UPFl gene, or 3) the yeast/human hybrid gene expressed from a MET25 promoter inserted into either a centromere (YCpRENTlCHI4-2) or a high copy plasmid ( YEpRENT 1 CHI4-2) . Methionine was omitted from the media to increase the expression of the hybrid gene (Perlick et al. 1996).
  • hybrid yeast/human UPFl gene prevented growth of these cells on -tip -met -leu -tyr media, demonstrating the ability of this protein to substitute for the yeast Upflp in preventing suppression of the leu2-2 and tyr7-l alleles (Fig. 6A).
  • the hybrid gene functioned better when expressed from a multicopy plasmid (Fig. 6A).
  • the expression of the chimeric protein had no effect on normal cell growth, since cells harboring these plasmids grew as well as wild-type on the -tip -met media (Fig. 6A).
  • Yeast/human UPFl gene promotes decay of nonsense-containing transcripts in yeast cells.
  • the abundance of the tyr7-l and leu2-2 nonsense-containing transcripts were determined in a upfl A strain harboring either the vector plasmid, the yeast UPFl gene, or the human yeast hybrid UPFl allele in a high copy plasmid.
  • Total RNAs from these cells were isolated and the abundances of the tyr7 and leu2 transcripts were analyzed by RNA blotting analysis, probing the blots with radiolabeled DNA probes encoding the TYR 7 and LEU2 genes (Weng et al. 1996a, Weng et al. 1996b).
  • the human Upflp interacts with the peptidyl release factors eRFl and eRF3:
  • the results described above demonstrate that the human homologue of the UPFl gene may also function in modulating the translation termination activity of the peptidyl release factors. Therefore, it was asked whether the full length rentl/hupfl would interact with eRFl and eRF3.
  • radiolabeled rentl/hupfl protein was synthesized in a coupled in vitro transcription/translation system. In vitro synthesis of the rentl/hupfl produced a band of approximately 130kD (Fig. 7 lane 1), consistent with the reported size of rentl/hupfl (Applequist et al. 1997).
  • the luciferase protein was also synthesized as described above and was used as a control protein for specificity of the interaction. Synthesis of the luciferase protein produced a 68kd protein (Fig. 7 lane 5). The rentl/hupfl or the luciferase protein was incubated with either GST, GST-eRFl or GST- eRF3 as described above and the interactions of rentl/hupfl or luciferase with these proteins were monitored by SDS-PAGE followed by autoradiography. The results demonstrated that the rentl/hupfl interacted with both the GST-eRFl or GST-eRF3 (Fig. 7 lane 3 and 4). The interaction was specific, since rentl/hupfl did not form a complex with GST protein (Fig.
  • the human gene contained amino and carboxyl terminal domains that were not present in the yeast UPFl gene, the human gene contained the cysteine-histidine- rich region and the helicase motifs found in the yeast homologue (Perlick et al., 1996; Applequist et al., 1997). Further, expression of a yeast/human hybrid of the UPFl genes functioned in a frameshift suppression assay when expressed in a upfl A strain (Perlick et al., 1996). The results presented here demonstrate that, analogous to the Upflp, expression of the yeast/human UPFl allele prevented the nonsense suppression phenotype observed in a upfl A strain harboring the nonsense-containing leu2-2 and tyr7-l alleles (Fig.
  • the termination event is a key point in the assembly of the surveillance complex and leads to enhanced translation termination and degradation of nonsense-containing transcripts.
  • Translation termination may also be an important event in regulating the stability or translation efficiency of wild-type transcripts.
  • the 3 '-untranslated regions of many transcripts encode regulatory elements that modulate the translation efficiency and/or stability of their respective mRNAs (reviewed in Ross, 1995; Jacobson and Peltz, 1996; Jacobson, 1996; Caponigro et al., 1995; Wickens et al., 1997). It is conceivable that the termination event is also the cue for the assembly of complexes that subsequently interact with the elements in the 3'-UTR that modulate their stability and/or translation efficiency.
  • PP2A protein phosphatase 2 A
  • eRFl translation termination factor
  • the PP2A may be then positioned in the appropriate location to modulate the activity of factors that regulate the translation efficiency or stability of the given transcripts.
  • this scenario is very similar to the how the NMD pathway function is perceived.
  • the basic premise for both wild-type and NMD is that termination is a rate limiting event that pauses the ribosome and signals the assembly of complexes that regulate subsequent events in the life span of a given transcript.
  • Example 2 The Upf3 Protein is a Component of the Surveillance Complex that Monitors both Translation and mRNA Turnover and Affects Viral Maintenance
  • the nonsense-mediated mRNA decay (NMD) pathway functions to degrade aberrant mRNAs which contain premature translation termination codons.
  • NMD mRNA decay
  • the Upfl, Upf2 and Up ⁇ proteins have been identified as /raws-acting factors involved in this pathway. Recent results have demonstrated that the Upf proteins may also be involved in maintaining the fidelity of several aspects of the translation process.
  • Certain mutations in the UPFl gene have been shown to affect the efficiency of translation termination at nonsense codons and/or the process of programmed -1 ribosomal frameshifting used by viruses to control their gene expression. Alteration of programmed frameshift efficiencies can affect virus assembly leading to reduced viral titers or elimination of the virus.
  • Up ⁇ protein functions to regulate programmed -1 frameshift efficiency.
  • a up ⁇ A strain demonstrates increased programmed -1 ribosomal frameshift efficiency which results in loss of ability to mantain the Mj virus.
  • the up ⁇ A strain is more sensitive to the antibiotic paromomycin than wild-type cells and frameshift efficiency increases in a up ⁇ A strain in the presence of this drug.
  • Up ⁇ p is epistatic to Upflp and Up ⁇ p. Based on these observations and the fact that the mo ⁇ -l allele of the UPFl gene also affects NMD and programmed -1 ribosomal frameshift efficiency, it was demonstrathed that the Up ⁇ proteins are part of a surveillance complex that functions to monitor translational fidelity and mRNA turnover.
  • Yeast media were prepared as described (Rose, M.D., Winston, F. and Hieter, P. (1990)). Yeast transformations were performed by the lithium acetate method (Schiestl, R.H., and Gietz, R.D. (1989)). Cytoductions of L-A and M, into rho-o strains were as described previously (Dinman, J.D., and Wickner, R.B. (1992)) using strains 3164 and 3165 (Dinman, J.D. and Wickner, R.B. (1994); Dinman, J.D., and Wickner, R.B. (1992)) as cytoduction donors, ⁇ -galactosidase ( ⁇ -gal) assays followed standard protocols (Guarente, L. (1983)).
  • T e killer assay was carried out as previously described (Dinman, J.D., and Wickner, R.B. (1992)) by replica plating colonies onto 4.7MB plates newly seeded with a lawn of 5x47 killer indicator cells (0.5 ml of a suspension at 1 unit of optical density at 550 nm per ml per plate). After 2 days at 20°C, killer activity was observed as a zone of growth inhibition around the killer colonies. To quantitate loss of killer activity, colonies that had been identified as killer + were re-streaked for single colonies and the percentage of killer colonies were determined.
  • TNA Total nucleic acids
  • RNA abundance of the lacZ -1 frameshift reporter mRNA and U3 snRNA was determined by ribonuclease protection assays essentially as described (Sambrook).
  • RNA probes were labelled with [ ⁇ - 32 P] UTP.
  • a pGEM derived plasmid containing the LacZ gene was digested with Hindi and in vitro transcribed with RNA polymerase T7.
  • pGEM-U3 a pGEM-derived plasmid, was cut with Sspl and in vitro transcribed with RNA polymerase T3.
  • L-A and M, (+) strand RNA probes were made as previously described using [ ⁇ - 32 P]CTP labeled T3 RNA polymerase runoff transcripts (28).
  • a up ⁇ A strain demonstrates an increased efficiency of programmed -1 ribosomal frameshifting: mof4-l is a unique allele of the UPFl gene that specifically increases programmed -1 ribosomal frameshifting efficiency and promotes loss of the M, satellite virus.
  • a upfl A strain does not demonstrate these phenotypes.
  • Other factors of the putative surveillance complex including the Upf2 or Up ⁇ proteins, also affect programmmed -1 ribosomal frameshifting. Therefore, isogenic strains harboring deletions of the UPF genes were investigated which demonstrated increased ribosomal frameshifting efficiencies.
  • Plasmid pTI25 serves as the 0-frame control since the lacZ is in the 0-frame with respect to the translational start site (Fig. 8).
  • plasmid pF8 an L-A derived programmed -1 ribosomal frameshift signal is cloned into the polylinker and the lacZ gene is in the -1 frame with respect to the translational start site (Fig. 8). Therefore, in this construct, the lacZ gene will be translated only if the ribosome shifts frame in the -1 direction.
  • the +1 frameshift reporter plasmid, pJD104 (Fig.
  • the -1 ribosomal frameshift efficiency (%) was determined by the ratio of ⁇ - galactosidase activity in a strain harboring the -1 ribosomal frameshifting reporter plasmid to the activity in the same strain harboring the 0 frame control plasmid.
  • 2 L- AHN and M were introduced into the strains by cytoduction and the maintenance (+) or loss (-) of M] dsRNA was analyzed by the killer plate assay and Northern blot analyses as described in Material and Methods.
  • the abundance of the frameshift reporter transcript is equivalent in the upf A strains:
  • the -1 frameshift reporter transcripts used in these assays have short protein coding regions 5' of the frameshift site followed by sequences that code for a reporter protein and that is out of frame with the translation initiation site of the 5' open reading frame.
  • the apparent changes in ribosomal frameshifting efficiencies could result from changes in the abundance of the LacZ -1 frameshift reporter mRNA which the translational machinery may recognize as a nonsense-containing mRNA. Deletion of the UPF genes could lead to stabilization of the -1 frameshift reporter transcript, resulting in increased synthesis of the ⁇ -gal reporter protein.
  • the abundance of the lacZ -1 frameshift reporter mRNA was determined by RNase protection analysis. As a loading control, it was determined the abundance of the U3 snRNA. Quantitation of the hybridizing bands revealed that the abundances of the lacZ frameshift reporter mRNA, normalized to the U3 snRNA, were equivalent in isogenic wild-type, upfl A, up ⁇ A and up ⁇ A strains (Fig. 9).
  • the M, killer virus is not maintained in a up ⁇ A strain: Changing the efficiency of -1 ribosomal frameshifting alters the ratio of Gag to Gag-pol proteins available for viral particle assembly, consequently interfering with viral propagation (Cui, Y., Dinman, J.D., and Peltz, S.W. (1996); Dinman, J.D. and Wickner, R.B. (1994); Dinman, J.D., and Wickner, R.B. (1992); Dinman, J.D., Ruiz-Echevarria, M.J., Czaplinski, K. and Peltz, S.W. (1997b)).
  • the L-A and M, viruses were introduced by cytoduction into isogenic wild-type UPF + , upfl A, up ⁇ A and up ⁇ A strains, and these cells were grown and replica plated onto a lawn of cells sensitive to the killer toxin. Cells maintaining the M j virus secrete the killer toxin, creating a ring of growth inhibition, whereas cells which have lost M ] do not demonstrate this growth inhibition (Cui, Y., Dinman, J.D., and Peltz, S.W. (1996); Dinman, J.D., and Wickner, R.B. (1992); Dinman, J.D., Ruiz-Echevarria, M.J., Czaplinski, K. and Peltz, S.W.
  • RNAs were transferred to nitrocellulose and hybridized with [ ⁇ - 3 P]CTP labeled L-A and M, (+) strand RNA specific probes. The results are shown in Fig. 10B.
  • the up ⁇ A strain demonstrates increased sensitivity to paromomycin: Strains harboring mutations that diminish translational fidelity are hypersensitive to the aminoglycoside antibiotic paromomycin, a drug that is thought to increase the frequency of misreading in yeast. Previous results demonstrated that cells harboring the mo -l allele of the UPFl gene, which increases the efficiency of -1 ribosomal frameshift, also showed an increased sensitivity to paromomycin than the isogenic wild-type strain. It was determined whether a up ⁇ A strain also demonstrates increased sensitivity to this antibiotic.
  • Paromomycin sensitivity was monitored in isogenic wild-type and up ⁇ A strains by placing a disc containing 1 mg of paromomycin onto a lawn of cells and determining the zone of growth inhibition around the disc (Fig. 11).
  • the results demonstrate that, analogous to a mo -l strain, a up ⁇ A strain was more sensitive to paromomycin than the isogenic wild-type strain. Neither the upfl A or up ⁇ A strains demonstrate hypersensitivity to paromomycin.
  • paromomycin on -1 ribosomal frameshifting was analyzed further by ⁇ - galactosidase assay using plasmids pF8 (-1 frameshift reporter construct) or pTI25 (zero frame control) in isogenic wild-type and up ⁇ A strains.
  • Cells were grown in liquid media in the presence of different concentrations of the drug and the ⁇ -galactosidase activity was determined, normalizing to thenumber of cells used in the assay.
  • the ⁇ -galactosidase activity from up ⁇ A cells carrying pF8 (-1 frameshift reporter construct) increased continuously with increased concentrations of paromomycin.
  • the programmed frameshifting and killer phenotypes of a up ⁇ A allele are independent of the other upf A alleles:
  • the epistatic relationships between upfl A, up ⁇ A and up ⁇ A were examined with regard to both -1 ribosomal frameshifting efficiencies and killer maintenance.
  • Both programmed -1 ribosomal frameshifting and killer phenotypes were monitored as described above in isogenic UPF ,upflA up ⁇ A, upfl A up ⁇ A, up ⁇ A up ⁇ A and upfl A up ⁇ A up ⁇ A strains. The results of these experiments are shown in Table 3.
  • the Upf proteins are part of the surveillance complex that monitors both mRNA turnover and translation.
  • the NMD pathway is an example of a mechanism that the cell has evolved to rid itself of aberrant nonsense-containing transcripts which, when translated, could produce anomalous peptides that can dominantly interfere with the normal cellular functions (Jacobson, A. and Peltz. S.W. (1996); Ruiz-Echevarria, M.J., K. Czaplinski, and Peltz, S.W. (1996); Weng, Y., M.J. Ruiz-Echevarria, S. Zhang, Y. Cui, K. Czaplinski, J. Dinman, and S.W. Peltz.
  • the mo -l allele of the UPFl gene demonstrates an increase in programmed -1 ribosomal frameshifting efficiency and is unable to mantain the M, killer virus (Cui, Y., Dinman, J.D., and Peltz, S.W. (1996)).
  • mo ⁇ -l mutants manifest increased programmed -1 ribosomal efficiency (Cui, Y., Dinman, J.D.D., Goss Kinzy, T. and Peltz, S.W. (1997)).
  • the mo ⁇ -l mutant is allelic to the SUI1 gene (Cui, Y., Dinman, J.D.D., Goss Kinzy, T. and Peltz, S.W.
  • the Up ⁇ p is the key factor that links the Up ⁇ complex to programmed -1 ribosomal frameshifting.
  • the increased programmed -1 ribosomal frameshifting in a up ⁇ A strain is not a consequence of stabilizing the reporter transcript to a greater degree than that observed in either upfl A or up ⁇ A strains (Fig. 9).
  • the results presented here indicate that the Up ⁇ p has a function in ensuring appropriate maintenance of translational reading frame.
  • the function of the Up ⁇ p in this process appears to be genetically epistatic to the Upflp and Up ⁇ p, since the programmed -1 frameshifting and killer maintenance phenotypes of a up ⁇ A are observed in upfl A and up ⁇ A strains (Table 3).
  • the results presented here demonstrate that the Up ⁇ 's may have distinct roles that can affect different aspects of the translation and mRNA turnover processes. Importantly these results may also have practical implications, since many viruses of clinical, veterinary and agricultural importance utilize programmed frameshifting (reviewed in Brierley, I.
  • mutations in the UPF genes can result in altered translation termination phenotypes, increased programmed frameshifting and stabilization of nonsense-containing transcripts (Weng, Y., K. Czaplinski, and Peltz, S.W. (1996a); Weng, Y., K. Czaplinski, and Peltz, S.W. (1996b); Cui, Y., Dinman, J.D., and Peltz, S.W. (1996).; reviewed in Ruiz-Echevarria, M.J., K. Czaplinski, and Peltz, S.W. (1996); Weng, Y., M.J. Ruiz-Echevarria, S. Zhang, Y. Cui, K.
  • a paused ribosome may be a key event that promotes assembly of the Up ⁇ complex, which can subsequently monitor these processes.
  • Both programmed frameshifting and translation termination involve a ribosomal pause (Wolin, S.L. and Walter, P. (1988); Tu, C, Tzeng, T.-H. and Bruenn, J.A. (1992); reviewed in Tate, W.P. and Brown, CM. (1992) ).
  • the results show that the interaction of the translation termination release factors eRFl and eRF3 with a paused ribosome containing a termination codon in the A site helps promote the assembly of the Up ⁇ complex.
  • RNA pseudoknot following the slippery site promotes a ribosomal pause (Tu, C, Tzeng, T.-H. and Bruenn, J.A. (1992);Somogyi, P., Jenner, A.J., Brierley, LA. and Inglis, S.C. (1993) ).
  • the paused ribosome may also trigger assembly of the surveillance complex.
  • This complex, or a subset of the Upf proteins, may help the ribosome to maintain the appropriate translational reading frame. In the absence of the these factors the ribosome is more prone to slip and change reading frame.
  • NAM7 nuclear gene encodes a novel member of a family of helicases with a Zl-6n-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae. J. Mol Biol. 224, 575- 587.
  • mo ⁇ -l is an allele of the UPF1/IFS2 gene which affects both mRNA turnover and -1 ribosomal frameshifting efficiency.
  • Peptidyl- transferase inhibitors have antiviral properties by altering programmed -1 ribosomal frameshifting efficiencies: development of model systems. Proc. Natl. Acad. Sci. USA 94, 6606-6611.
  • Eukaryotic polypeptide chain release factor eRF3 is an eRFl- and ribosome-dependent guanosine triphosphatase. RNA 4, 334-341.
  • Nonsense suppressors partially revert the decrease of the mRNA levels of a nonsense mutant allele in yeast. Curr. Genetics 77, 77-79.
  • mRNA destabilization triggered by premature translational termination depends on three mRNA sequence elements and at least one trans-acting factor. Genes & Dev. 7. 1737-1754.
  • ATP is a cofactor of the Upfl protein that modulates it translation termination and RNA binding activities.
  • Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRFl and eRF3. EMBO J. 14, 4065-4072.

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EP99953357A 1998-05-28 1999-05-27 Procede de modulation de l'efficience de terminaison de translation et de la degradation de l'arnm aberrant faisant intervenir un complexe de surveillance comprenant la proteine humaine upf1p, le facteur 1 et le facteur 3 de liberation eucaryote Expired - Lifetime EP1102838B1 (fr)

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US20030032158A1 (en) * 1998-05-28 2003-02-13 University Of Medicine And Dentistry Of New Jersey Method of modulating the efficiency of translation termination and degradation of aberrant mRNA involving a surveillance complex comprising human Upf1p,eucaryotic release factor 1 and eucaryotic release factor 3
ATE367399T1 (de) * 1998-07-22 2007-08-15 Univ New Jersey Med Rna helicasen die die termination der translation beeinflussen
US6630294B1 (en) 1998-07-22 2003-10-07 University Of Medicine And Dentistry Of New Jersey Subfamily of RNA helicases which are modulators of the fidelity of translation termination and uses thereof
US6458538B1 (en) * 1999-12-14 2002-10-01 Ptc Therapeutics, Inc. Methods of assaying for compounds that inhibit premature translation termination and nonsense-mediated RNA decay
US20040161765A1 (en) * 2001-04-13 2004-08-19 Dietz Harry C. Methods and compositions for identifying disease genes using nonsense-mediated decay inhibition
US20040072201A1 (en) * 2002-04-16 2004-04-15 Dietz Harry C. Methods of identifying compounds that inhibit nonstop degradation of mRNA
US7291461B2 (en) * 2002-06-21 2007-11-06 Ptc Therapeutics, Inc. Methods for identifying small molecules that modulate premature translation termination and nonsense mRNA decay
WO2004037976A2 (fr) * 2002-08-22 2004-05-06 University Of Rochester Mecanisme de controle de qualite de l'arnm nmd
WO2005086768A2 (fr) * 2004-03-11 2005-09-22 Albert Einstein College Of Medicine Of Yeshiva University Production amelioree de proteines fonctionnelles a partir de genes defectueux
EP2251437B1 (fr) * 2009-05-13 2013-12-04 Hannelore Breitenbach-Koller Procédé d'identification de composés qui contrôlent l'activité translationnelle des protéines ribosomales dans l'expression mRNA différentielle
CN113908589B (zh) * 2021-10-08 2022-09-27 天津工业大学 一种表面印迹抗体的疏水电荷诱导模式膜层析介质及其制备方法
KR102864696B1 (ko) * 2021-11-08 2025-09-29 한양대학교 산학협력단 Upf1 단백질 또는 upf1 단백질 유래 폴리펩티드의 암의 예방 또는 치료 용도

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US5874231A (en) 1994-08-22 1999-02-23 Mcgill University Methods of screening for non-hormone compounds which effect modulation of polypeptide translation
US5679566A (en) 1995-01-20 1997-10-21 University Of Massachusetts Medical Center Yeast NMD2 gene
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US6486305B1 (en) 2002-11-26
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WO1999061600A9 (fr) 2000-07-06
JP4889150B2 (ja) 2012-03-07
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